Of the Box

Lab-grown ‘minibrains’ are revealing what makes humans special

Ever since Alex Pollen was a boy talking with his neuroscientist father, he wanted to know how evolution made the human brain so special. Our brains are bigger, relative to body size, than other animals’, but it’s not just size that matters. “Elephants and whales have bigger brains,” notes Pollen, now a neuroscientist himself at the University of California, San Francisco. Comparing anatomy or even genomes of humans and other animals reveals little about the genetic and developmental changes that sent our brains down such a different path.

Geneticists have identified a few key differences in the genes of humans and apes, such as a version of the gene FOXP2 that allows humans to form words. But specifically how human variants of such genes shape our brain in development—and how they drove its evolution—have remained largely mysterious. “We’ve been a bit frustrated working so many years with the traditional tools,” says neurogeneticist Simon Fisher, director of the Max Planck Institute for Psycholinguistics in Nijmegen, the Netherlands, who studies FOXP2.

Now, researchers are deploying new tools to understand the molecular mechanisms behind the unique features of our brain. At a symposium at The American Society of Human Genetics here last month, they reported zooming in on the genes expressed in a single brain cell, as well as panning out to understand how genes foster connections among far-flung brain regions. Pollen and others also are experimenting with brain “organoids,” tiny structured blobs of lab-grown tissue, to detail the molecular mechanisms that govern the folding and growth of the embryonic human brain. “We used to be just limited to looking at sequence data and cataloging differences from other primates,” says Fisher, who helped organize the session. “Now, we have these exciting new tools that are helping us to understand which genes are important.”

Most of the talks focused on the development of the cerebral cortex, the wrinkled outer layer of the brain that orchestrates higher cognitive functions such as memory, attention, awareness, language, and thought. The human cortex is special, with three times as many cells as that of chimps, and deeper folds that help pack in those extra cells. These differences begin to unfold in the earliest phase of fetal development, but researchers know little about the genes that direct this transformation and the molecules they encode.

In his talk, Wieland Huttner, a molecular cell biologist and developmental neurobiologist at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany, explained how his team searched databases for proteins and other gene products expressed in the human brain in these earliest phases of development. They zeroed in on three proteins found in the extracellular matrix that surrounds developing cells in fetal brain tissue. When they added these proteins to cultures of brain tissue from aborted human fetuses, the tissue formed folds, as it does in human fetuses at about 20 weeks of gestation.

What’s more, MPI-CBG postdoc Katie Long noticed that the three proteins formed folds only after they clustered with another complex glycan molecule called hyaluronic acid. This complex molecule has many functions, such as carrying signals between cells and promoting cell growth, which is why it’s used in face creams. Although researchers knew that hyaluronic acid shows up in neural tissue, they did not know it played such a critical role in human brain development. “They have identified key molecules that facilitate cortical folding,” says neuroscientist Louis Reichardt, director of the Simons Foundation Autism Research Initiative in New York City, who heard the talk.

Working with paleogeneticist Svante Pääbo and biophysical chemist Barbara Treutlein, both of the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, Huttner’s team is growing brain organoids, tiny bits of tissue that develop in culture in a way that resembles an embryonic brain. The researchers coaxed white blood cells from humans and other apes into forming stem cells, from which they grew organoids. The organoids grow for several weeks—sometimes up to a year—allowing the researchers to compare growth and pinpoint where the differences among species arise. “Organoids are very powerful because you cannot get fetal chimp brain tissue,” because they are a threatened species, Huttner says.

The organoids from great apes and people grew in remarkably similar ways. All formed the same types of stem cells, which give rise to “progenitor” cells that, in turn, divide into neurons and eventually organize themselves in six layers of brain tissue. But when the researchers used live microscopy to watch how the 4-millimeter-wide organoids developed, they noticed that human progenitor cells took 50% longer than those of the other great apes to arrange their chromosomes before splitting into daughter cells. The human cells seemed to invest much more time in the phase of cell division called metaphase. Somehow, this lengthening of metaphase early in development appears to cause more progenitor cells to be made later, Huttner says.

Other researchers are trying to unravel the web of connectivity of the human brain. Neuroscientist Fenna Krienen of Harvard Medical School in Boston used functional MRI (fMRI) in 1000 people to show that human brains make synaptic connections across vast distances in the cerebral cortex. Rodent neurons, in contrast, limit their connections to nearby areas. Krienen and her then–Ph.D. adviser, Randy Buckner at Harvard, hypothesized that as the human cortex expanded in the course of evolution, it reorganized to allow more complex connections between regions.

Since then, Krienen has been making an inventory of the cells found in particular layers of the neocortex. Last year she and her colleagues linked the fMRI results to genetics in the brain tissue of six adults. They reported in the Proceedings of the National Academy of Sciences that 19 genes were turned on in the same underlying areas as the cortical connections revealed by fMRI. In mice, those genes are expressed earlier in development, in other cortical layers. It seems that sometime over the course of primate and human evolution, these genes were directed to become active later in development.

Now, Krienen is using a new single-cell analysis method developed in Steve McCarroll’s lab at Harvard (where she is now a postdoc) to refine these results from layers of tissue to single cells. She’s looking at all the genes—including the original 19 and many more—expressed by each cell in the connected regions. She hopes to pinpoint which genes are expressed in each cell type when brain cells make long distance connections, and to make similar maps in other primates to chart what changed as brains rewired over the course of evolution.

Aside from their evolutionary importance, these studies have implications for research on mental disorders. As connections proliferated across the brain, more opportunities arose for missed connections. Autism and other specific mental disorders may be caused in part by “specific circuits or regions of the brain [that] had problems with connectivity,” Krienen says.

“These new technologies are simply spectacular,” says Reichardt, speaking of the variety of approaches detailed in the session. “We’re learning a heck of a lot.”

It’s still early days for this budding field, but it’s already clear that more than one or two genes sculpted the uniquely human brain, says symposium co-organizer Evan Eichler, a geneticist at the University of Washington in Seattle. “It’s a concert of dozens of human events that culminated in this amazing organ,” he says.